DNA encoding GLS1

ABSTRACT

The DNA encoding glucan synthesis gene 1 (GLS1) is cloned and used in an in vitro assay to screen for compounds that modulate 1,3 β-D glucan synthase activity.

CROSS RELATED TO OTHER APPLICATIONS

This is a continuation of U.S. Ser. No. 08/249,420 filed May 26, 1994,now U.S. Pat. No. 5,484,724.

BACKGROUND OF THE INVENTION

A DNA molecule containing a gene which reverses a mutant phenotype of astrain of Saccharomyces cerevisiae is isolated and purified. The gene isGLS1 (glucan synthesis gene 1). GLS1 encodes a subunit of 1,3-β-D glucansynthase. The protein encoded by GLS1 represents a target for drugtherapy for fungal disease. The invention includes homologues of GLS1 isisolated from other fungi, such as Aspergillus fumigatus, Candidaalbicans, Schizosaccharmomyces pombe and Phytophthora infestans.

Understanding the mode of action of therapeutic compounds requires avariety of experimental approaches. One approach involves the isolationof organisms resistant or sensitive to test compounds. Such organismsmay be used to isolate genes encoding the drug targets.

The fungal cell wall is a complex structure composed of a number ofpolymers: chitin, α- and β-glucans, and mannoproteins. The fungal cellwall is involved in a variety of vital cellular processes: vegetativegrowth, morphogenesis, uptake and secretion of macromolecules andprotection against osmotic changes are affected by changes in thecomposition and integrity of the cell wall. Antifungal compounds whichact via the inhibition of cell wall synthesis (a process essential tofungi and absent from mammalian cells) may have high fungicidal activityand low toxicity to mammalian cells.

One class of β-glucan inhibitors is comprised of lipopeptide antibioticssuch as aculeacin A, echinocandin B and the pneumocandins. Thesecompounds are cyclic hexapeptides that contain a non-polar fatty acidside chain. Echinocandins are fungicidal because they inhibit synthesisof 1,3-β-D glucan, which disrupts the integrity of the cell wall andcauses lysis of yeast cells. In vitro echinocandins inhibitpolymerization of glucose into 1,3-β-D glucan.

Another class of β-glucan synthesis inhibitors comprises thepapulacandins and chaetiacandin. These compounds contain a glycosidecomponent connected to an aromatic ring system and two long chain fattyacids. These compounds have the same mode of action as theechinocandins.

It has been shown that Pneumocystis carinii has β-glucan in the wall ofits cyst form (Matsumoto, Y., et al., 1989, J. Protozool. 36: 21S-22S).Inhibitors of β-glucan synthesis, such as papulacandins andechinocandins, may be useful in the treatment of P. carinii infections.In a rat model of P. carinii pneumonia, L-671,329 (an echinocandin) andL-687,781 (a papulacandin) were both effective in reducing the number ofcysts in the lungs of infected rats (D. M. Schmatz et al., 1990, PNAS87: 5950-5954). These results suggest that β-glucan synthesis is atarget for the identification of therapeutics useful in the treatment ofP. carinii infections.

There have been a number of efforts to isolate drug-resistant yeaststrains affected in β-glucan synthesis. The mutants that have beenisolated include acul (Mason, M. M., et al., 1989, Cold Spring HarborLaboratory, Abstract #154), and pap1 (Duran, A., et al., 1992, Profilesin Biotechnology (T. G. Villa and J. Abalde, Eds.) Serivicio dePublicaciones, Universidad de Santiago, Spain. pp. 221-232).

In the present work a more potent echinocandin (L-733,560) was used as aselective agent to isolate mutant strains specifically affected inglucan synthesis. One mutant (strain MS14) is echinocandin-resistant andis also supersensitive to the chitin synthase inhibitor nikkomycin Z.The mutation in MS14 maps to the FKS1 gene and is designated fks1-4.Another mutant (strain MS1) is resistant to echinocandins andsupersensitive to both papulacandin and rapamycin. Strain MS1 was usedto clone the GLS1 gene.

SUMMARY OF THE INVENTION

A DNA molecule encoding a protein involved in biosynthesis of 1,3-β-Dglucan (GLS1) is identified, cloned, expressed and used in assays toscreen for antifungal compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Segregation pattern (2:2) of meiotic products indicating singlegene mutations conferring resistant to echinocandins. Cells representingthe 4 meiotic products of tetrads resulting from a crosses between MS1and the wild-type strain GG100-14D were spotted on media containing 7.5μM of L,733-560. Following growth at 28° C. for two days, two of foursegregants were able to grow indicating the 2:2 segregation of thedrug-resistance phenotype.

FIG. 2. Strain MS1 (gls1-1 mutant) was tested for resistance to theechinocandin L-733,560 by the broth microdilution assay.

FIG. 3. Morphological defects in strain MS1 (gls1-1 mutant) showingaggregates of cells (A) that start to lyse towards the center of theaggregate (B) before all the cells in the aggregate lyse (C&D).

FIG. 4. Effect of L-733,560 and Nikkomycin Z on 1,3-β-D glucan synthesisand chitin synthesis. Membrane extracts prepared from X2180-1A(wild-type) and from MS1 (gls1-1 mutant) were used to catalyze1,3-β-glucan synthase reactions (A) and chitin synthase reactions (B).UDP-glucose (A) and N-acetylglucosamine (B) were used as substrates.

FIG. 5. Effect of GTPγS on 1,3-β-glucan synthase activity. Membraneextracts from wild-type and mutant strains were used to prime1,3-β-glucan synthase reactions in the presence of increasingconcentrations of GTPγS.

FIG. 6. Localization of the gls1-1 minimum complementing fragment. Apartial restriction map of the 17-kb clone containing gls1-1complementing activity is depicted (A). The direction of transcriptionof GLS1 is indicated by the arrow. The yeast genomic DNA fragmentsrepresented by the lines below the restriction map (B) were insertedinto the centromeric plasmid YCP50. The recombinant plasmids weretransformed into MS1 (gls1-1 mutant). Transformed cells were tested forcomplementation of the echinocandin-resistance phenotype. The plasmidsas represented from the top to the bottom are pJAC2, pHF, pEF.Abbreviations for restriction enzymes: D, DpnI; E, EcoRI; H, HindIII; K,KpnI.

FIG. 7. GLS1 mediates sensitivity to echinocandins.

FIG. 8. Hydropathy plot of the 348 amino acid GLS1 gene product.

FIG. 9. Nucleotide and predicted amino acid sequences of the S.cerevisiae GLS1. The nucleotide sequence is SEQ ID NO: 1 and the aminoacid sequence is SEQ ID NO: 2.

FIG. 10. GLS1 complements the papulacandin- and therapamycin-supersensitivity phenotypes of the gls1-1 mutation.

(A) Effect of rapamycin on cells carrying the gls1-1 mutation.

(B) Effect of papulacandin B on the same mutant cells. The mutant cellstransformed with GLS1 on a plasmid reverse sensitivity to both drugs.

FIG. 11. Southern hybridization of genomic DNA. To test whetherhomologues of the cloned GLS1 exist in other fungi, DNA probes from theGLS1 gene of S. cerevisiae were hybridized to genomic DNA from severalheterologous species. The existence of GLS1 homologues in severalspecies, including Candida albicans, Aspergillus fumigatus, Pneumocystscarinii and Schizosaccharomyces pombe is shown.

DETAILED DESCRIPTION OF THE INVENTION

A DNA molecule encoding a protein involved in biosynthesis of 1,3-β-Dglucan (GLS1) is identified, cloned, expressed and used in assays toscreen for antifungal compounds.

Antifungal compounds are used for treatment of fungal infections inanimals, including humans. There is increasing demand for safer and moreeffective antifungal compounds. Because the structure of the fungal cellwall differs from the structure of mammalian cell membranes, compoundsthat specifically interfere with the maintenance or biosynthesis of thefungal cell wall are targets of drug screens.

Cell wall biosynthesis is fundamental to the maintenance and growth offungi and plants. Cell walls provide skeletal support and mechanicalprotection of the protoplasts from the environment. Functions such asthe selective uptake of macromolecules, osmoregulation, cell growth andcell division occur in the cell wall. Enzymatic activities related tohydrolysis of extracellular nutrients and turnover of the cell wallmacromolecules during morphogenesis are associated with theextracellular matrix.

Polysaccharides account for as much as 80-90% of the cell wall of S.cerevisiae. The major cell wall polymers are glucan and mannan; inaddition, small amounts of chitin are present (Cabib, E. 1991,Antimicrob. Agents Chemother. 35: 170-173). It is believed that glucansupports and maintains rigidity of the cell wall while mannoproteinsregulate its permeability (Zlotnik et al., 1984).

Three types of glucan account for 30-60% of the cell wall of S.cerevisiae (Fleet, G. H., 1985, p. 24-56. In M. R. McGinnis (ed.),Current Topics in Medical Mycology Vol. I. Springer, Verlag, New York).The major form of glucan (60% of the total) is insoluble in alkali oracetic acid, is a branched 1-3-βpolymer, has fibrillar structurescontaining 3% of 1-6-βinterchain linkages, and lacks 1-6-βinterresiduelinkages. A second form of glucan (32% of the total) is soluble indilute alkali, has an amorphous structure and contains mainly1-3-βlinkages with some 1-6-βlinkages. A minor form of glucan (8% of thetotal) is acid-soluble, highly-branched and contains mainly1-6-βlinkages.

Echinocandins interfere with cell wall biosynthesis, most likely byinhibiting the synthesis of 1,3-β-glucan. A key enzyme in the cell wallformation is the 1,3-βglucan synthase. This enzyme is absent from animalcells, making it a target for development of antifungal compounds.1,3-βglucan synthase is a membrane-associated enzyme that usesUDP-glucose as a substrate and is stimulated by a detergent-solubleGTP-binding protein (Kang, M. S. and E. Cabib. Proc. Natl. Acad. Sci.USA 83: 5808-5812).

The techniques used to isolate drug-resistant mutants are similar tothose used to isololate auxotrophic, temperature-sensitive, andUV-sensitive mutants, such as described (Sherman et al., 1986).

The GLS1 gene may be isolated from a chromosomal DNA library bycomplementation of a mutation (gls1-1) which renders cells resistant toechinocandins (Sherman et al., 1986). The GLS1 gene may be isolated fromchromosomal DNA by preparing a library of DNA fragments in a DNA cloningvector and screening individual clones for the presence of GLS1. Forexample, a library of S. cerevisiae genomic DNA from strain GRF88 in theplasmid YCp50 can be obtained from the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852, as ATCC 37415.

A plasmid library may be prepared by isolating chromosomal DNA from purecultures of the microorganisms. The chromosomal DNA is fragmented forexample, by partial digestion with one or more restriction endonucleaseenzymes. The resulting DNA fragments are separated by size and may thenfragments are inserted into a cloning vector.

The cloning vector is cut with at least one restriction endonuclease,treated with phosphatase, and the DNA fragments are ligated with a DNAligase. The cloning vectors are used to transform host cells competentfor the uptake of DNA. Host cells for cloning, DNA processing, andexpression include but are not limited to bacteria, yeast, fungi, insectcells and mammalian cells. Escherichia coli K-12 strains RR1, HB101,JM109, DH11S, or DH5a are useful host cells. When about 5×10⁴independent genomic DNA fragments are ligated into a cloning vector, alibrary is formed. A complete library is likely to contain arepresentation of the entire genome. Competent host cells which take upand stably maintain a recombinant DNA molecule in the transformationprocedure can be identified by their ability to grow on mediumsupplemented with a plasmid-selective drug. For plasmid vectorscontaining the ampicillin resistance gene, ampicillin is the selectivedrug. To obtain full representation of a library, transformationmixtures are spread on agar plates and incubated under appropriateconditions. Transformed cells are resuspended from the surface of agarplates in a small volume of liquid medium. The cell suspension is usedto inoculate a larger volume of liquid medium supplemented with theselective drug, and incubated overnight at 37° C. Plasmid DNA is thenextracted from the cells by methods known in the art.

Screens to identify the GLS1 gene in the plasmid library can be devised.One strategy requires the use of a gls1-1 mutant of S. cerevisiae, suchas strain D2-8B or strain D2-8D. Cells are made competent to take up DNAand then transformed with library DNA. Transformants bearing the GLS1gene will exhibit a plasmid-dependent increase in sensitivity to aselective echinocandin.

Aliquots of the transformation mixture are plated on selective media.Colonies of transformants may be collected, resuspended in liquidmedium, pooled, and stored frozen at -80° C. in medium supplemented with25% glycerol. The titer, defined as the number of colony forming unitsper milliliter, is determined by methods known in the art.

Identification of transformants that contain the GLS1 gene isaccomplished by plating the library onto agar plates containingplasmid-selective medium such that a countable number of colonies growon each plate. A portion of each colony is transferred to two agarplates by replica plating: one plate contains plasmid-selective mediumsupplemented with a concentration of the selective echinocandin whichkills the cells with intermediate sensitivity, and a second platecontains plasmid-selective medium only. Positive clones grow normally onthe plate without echinocandin but grow poorly or not at all on theechinocandin-containing plate.

The echinocandin-sensitive phenotype of potential clones may be detectedby a variety of tests. In one test, cells from a colony are patcheddirectly onto the surface of plates containing different concentrationsof the selective echinocandin. The test is scored after two days ofincubation. Cells that grow poorly in the presence of the drug arepotential positives and are likely to contain plasmids carrying thecomplementary gene.

In a second test, a portion of each colony is transferred by replicaplating to an agar plate containing the selective echinocandin at aconcentration approximately twice that used in the first test. Positiveclones (clones that are sensitive to echinocandin) do not grow on theseplates.

In a third test, cells from a colony are inoculated intoplasmid-selective liquid medium and grown to saturation. An aliquot ofthe saturated culture is used to inoculate fresh liquid mediumsupplemented with or without the selective echinocandin. Growth ismeasured by optical density at a wavelength of 600 nm. Colonies that donot grow in the presence of echinocandin are scoredechinocandin-sensitive.

In another test, clones are tested in a broth microdilution assay,wherein a range of concentrations of the selective echinocandin aretested. Positive clones are more sensitive to the selective echinocandinthan the original resistant mutant.

Tests such as those described above may be used to screen a library ofgenomic DNA so as to identify a recombinant plasmid that contains afunctional copy of the GLS1 gene. To determine whether an increase insensitivity to echinocandin is due to a plasmid-encoded copy of GLS1,positive clones are cured of plasmid DNA and tested for a decrease insensitivity to echinocandin. If increased echinocandin sensitivity isdue to the presence of the plasmid, then plasmid loss results in theloss of this phenotype.

More direct proof that an increase in sensitivity to echinocandin is dueto the presence of a plasmid containing the GLS1 gene may be obtained byisolating plasmid DNA from a positive clone. Cells of E. coli competentto take up DNA are transformed with the plasmid, and transformants areidentified and isolated. Plasmid DNA is isolated from the transformed E.coli and then digested with restriction endonucleases to yield fragmentsof discrete sizes. The size of each fragment is estimated byconventional methods, such as gel electrophoresis. By digesting theplasmid with a variety of enzymes, a cleavage map is generated. Thecleavage map is distinct and specific for the cloned fragment. Adetailed cleavage map is sufficient to identify a particular gene withinthe genome. Fragments of the cloned gene, generated by digestion withendonucleases, may be purified from agarose gels and ligated intovectors suitable for sequencing by methods known in the art. Vectorsinclude, but are not limited to pUC18, pUC19, YEp24, pGEM3Zf(+),pGEM5Zf(+), and pGEM7Zf(-).

The GLS1 gene of S. cerevisiae may be used to isolate and characterizehomologous genes in pathogenic fungi. Because other fungi, which includebut are not limited to strains of C. neoformans, C. albicans, A.fumigatus, and Phytophthora infestans have 1,3-β-D glucan in their cellwalls, it is likely that a functional homologue of GLS1 exists in eachof these fungi. Functional homologues of GLS1 may exist in otherorganisms that have 1,3-β-D glucans in their cell walls.

GLS1 homologues may be detected by isolating chromosomal DNA from a testorganism. A portion of the isolated chromosomal DNA is cut with a numberof restriction enzymes. The digested fragments of DNA are separated bygel electrophoresis. The fragments are then transferred to a solidmembrane support. The membrane is then hybridized overnight with alabeled probe. The blot is washed and then exposed to XAR-5 film anddeveloped by conventional methods (Laskey and Mills (1977) FEBS Letters,82: 314-316). The conditions for washing the blot are such that only DNAfragments with a high degree of homology (estimated at ≧80%) willhybridize to the probe. The size and pattern of the digested fragmentswhich hybridize with the probe generate a genomic map. For eachorganism, the map is sufficient to specifically identify the GLS1homologue in the chromosome.

Mutations of the GLS1 gene, including but not limited to gls1-1 (strainMS1), gls1-2 (strain MS41) and gls1-3 (strain MS43), or disruptions ordeletions of GLS1, are useful for screening for glucan synthaseinhibitors. Such a screen relies on the increase inechinocandin-resistance and in papulacandin sensitivity of such mutantscompared to an GLS1 wild-type strain. Any technique capable of detectingthis difference in sensitivity can be used. A zone of inhibition assayon agar plates is particularly useful.

Cloned GLS1 cDNA may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce recombinant GLS1.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria, yeast,bluegreen algae, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector may contain: an origin of replication forautonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant GLS1 in mammalian cells. Commercially-available mammalianexpression vectors which may be suitable for recombinant GLS1expression, include but are not limited to, pMClneo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2)(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), andIZD35 (ATCC 37565).

DNA encoding GLS1 may also be cloned into an expression vector forexpression in a recombinant host cell. Recombinant host cells may beprokaryotic or eukaryotic, including but not limited to bacteria, yeast,mammalian cells and insect cells. Cell lines derived from mammalianspecies which may be suitable and which are commercially available,include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, infection, protoplast fusion, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce GLS1 protein.Identification of GLS1 expressing host cell clones may be done byseveral means, including but not limited to immunological reactivitywith anti-GLS1 antibodies, and the presence of host cell-associated GLS1activity.

Expression of GLS1 cDNA may also be performed using in vitro producedsynthetic mRNA. Synthetic MRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes.

To determine the GLS1 cDNA sequence(s) that yields optimal levels ofenzymatic activity and/or GLS1 protein, modifed GLS1 cDNA molecules areconstructed. Host cells are transformed with the cDNA molecules and thelevels of GLS1 RNA and protein are measured.

Levels of GLS1 protein in host cells are quantitated by a variety ofmethods such as immunoaffinity and/or ligand affinity techniques.GLS1-specific affinity beads or GLS1-specific antibodies are used toisolate ³⁵ S-methionine labelled or unlabelled GLS1 protein. LabelledGLS1 protein is analyzed by SDS-PAGE. Unlabelled GLS1 protein isdetected by Western blotting, ELISA or RIA assays employing GLS1specific antibodies.

Following expression of GLS1 in a recombinant host cell, GLS1 proteinmay be recovered to provide GLS1 in active form. Several GLS1purification procedures are available and suitable for use. RecombinantGLS1 may be purified from cell lysates or from conditioned culturemedia, by various combinations of, or individual application offractionation, or chromatography steps that are known in the art.

In addition, recombinant GLS1 can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full-length nascent GLS1 orpolypeptide fragments of GLS1.

The recombinant protein may be used to generate antibodies. The term"antibody" as used herein includes both polyclonal and monoclonalantibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2fragments that are capable of binding antigen or hapten.

Monospecific antibodies to GLS1 are purified from mammalian antiseracontaining antibodies reactive against GLS1 or are prepared asmonoclonal antibodies reactive with GLS1 using standard techniques.Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for GLS1. Homogenous binding as used herein refers tothe ability of the antibody species to bind to a specific antigen orepitope, such as those associated with the GLS1, as described above.Enzyme-specific antibodies are raised by immunizing animals such asmice, rats, guinea pigs, rabbits, goats, horses and the like, withrabbits being preferred, with an appropriate concentration of GLS1either with or without an immune adjuvant.

Monoclonal antibodies (mAb) reactive with GLS1 may be prepared byconventional methods, such as by immunizing inbred mice with GLS1.

In vitro production of anti-GLS1 is carried out by growing the hydridomain DMEM containing about 2% fetal calf serum to obtain sufficientquantities of the specific mAb. The mAb are purified by techniques knownin the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of GLS1 inbody fluids or tissue and cell extracts.

Methods such as those described above may be used to producemonospecific antibodies may be utilized to produce antibodies specificfor GLS1 polypeptide fragments or full-length nascent GLS1 polypeptide.

Kits containing GLS1 cDNA, antibodies to GLS1 or GLS1 protein may beprepared. Such kits are used to detect DNA which hybridizes to GLS1 DNAor to detect the presence of GLS1 protein or peptide fragments in asample. Such characterization is useful for a variety of purposesincluding but not limited to forensic analyses, taxonomic determinationsand epidemiological studies.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of GLS1DNA, GLS1 RNA or GLS1 protein. The recombinant proteins, DNA molecules,RNA molecules and antibodies lend themselves to the formulation of kitssuitable for the detection and typing of GLS1. Such a kit would comprisea compartmentalized carrier suitable to hold in close confinement atleast one container. The carrier would further comprise reagents such asrecombinant GLS1 protein or anti-GLS1 antibodies suitable for detectingGLS1. The carrier may also contain means for detection such as labeledantigen or enzyme substrates or the like.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the GLS1 sequence butwill be capable of hybridizing to GLS1 DNA even in the presence of DNAoligonucleotides with mismatches. The mismatched DNA oligonucleotidesmay still hybridize to the GLS1 DNA to permit identification andisolation of GLS1 encoding DNA.

DNA encoding GLS1 from a particular organism may be used to isolate andpurify homologues of GLS1 from other organisms. To accomplish this, thefirst GLS1 DNA may be mixed with a sample containing DNA encodinghomologues of GLS1 under appropriate hybridization conditions. Thehybridized DNA complex may be isolated and the DNA encoding thehomologous DNA may be purified therefrom.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences which containalternative codons which code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally-occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate.

As used herein, a "functional derivative" of GLS1 is a compound thatpossesses a biological activity (either functional or structural) thatis substantially similar to the biological activity of GLS1. The term"functional derivatives" is intended to include the "fragments,""variants," "degenerate variants," "analogs" and "homologs" or to"chemical derivatives" of GLS1. The term "fragment" is meant to refer toany polypeptide subset of GLS1. The term "variant" is meant to refer toa molecule substantially similar in structure and function to either theentire GLS1 molecule or to a fragment thereof. A molecule is"substantially similar" to GLS1 if both molecules have substantiallysimilar structures or if both molecules possess similar biologicalactivity. Therefore, if the two molecules possess substantially similaractivity, they are considered to be variants even if the structure ofone of the molecules is not found in the other or even if the two aminoacid sequences are not identical. The term "analog" refers to a moleculesubstantially similar in function to either the entire GLS1 molecule orto a fragment thereof.

The present invention is also directed to methods for screening forcompounds which modulate that expression of DNA or RNA encoding GLS1 aswell as the function of GLS1 protein in vivo. Compounds which modulatethese activities may be DNA, RNA, peptides, proteins, ornon-proteinaceous organic molecules. Compounds may modulate byincreasing or attenuating the expression of DNA or RNA encoding GLS1 orthe function of GLS1 protein. Compounds that modulate the expression ofDNA or RNA encoding GLS1 or the function of GLS1 protein may be detectedby a variety of assays. The assay may be a simple "yes/no" assay todetermine whether there is a change in expression or function. The assaymay be made quantitative by comparing the expression or function of atest sample with the levels of expression or function in a standardsample.

The following examples are provided to further define the inventionwithout, however, limiting the invention to the particulars of theseexamples.

EXAMPLE 1

Strains Plasmids and Media

The yeast strains used in this study are listed in Table 1. The mutantstrains are derived from strain X2180-1A.

                  TABLE 1    ______________________________________    Strain       Relevant Properties    ______________________________________    X2180-1A     MATa GLS1 (wt, Ech.sup.S)    MS1          MATa gls1-1 (Ech.sup.R)    MS41         MATa gls1-2 (Ech.sup.R)    MS7-43       MATa gls1-3 (Ech.sup.R)    GG100-14D    MATα GLS1 (wt, Ech.sup.S)    D2           MATa/MATα (MS41 × GG100-14D)    D12          MATa/MATα (MS1 × GG100-14D)    D28          MATa/MATα (MS7-43 × GG100-14D)    D132         MATa/MATα (D28-18C × D28-3B)    D136         MATa/MATα (D28-18C × D2-1D)    D137         MATa/MATα (D28-18C × D12-11D)    D140         MATa/MATα (D28-9D × D2-1B)    D141         MATa/MATα (D12-7D × D2-1B)    D142         MATa/MATα (D2-2D × D2-1B)    D2-1B        MATα ura3-52 gls1-2    D2-1D        MATα ura3-52 gls1-2    D2-2D        MATα his3 gls1-2    D12-7D       MATα his3 gls1-1    D12-11D      MATα ura3-52 gls1-3    D28-3B       MATα his3 gls1-3    D28-9D       MATα his3 gls1-3    D28-18C      MATα ura3-52 his3 gls1-3    D2-5A        MATα his3 ura3-52 GLS1    MS100        MATα his3 trpl glsl::URA3-52    MS101        MATα hiS3 gls1::URA3-52    ______________________________________

Strains GG100-14D and X2180-1A were obtained from K. Bostian and C.Ballou respectively. The mutants were generated in X2180-1A andoutcrossed to GG100-14D. Abbreviations: wt, wild-type; Ech,echinocandin; S, sensitive; R, resistance.

    ______________________________________                               Source of    Plasmid   Description      cloned DNA    ______________________________________    pJAC2     16-Kb GLS 1 clone in YCp50                               GRF88    pJAC1      4-kb GLS 1 clone in YCp50                               GRF88    pJAC4      4-Kb GLS 1 clone in YEP24                               GRF88    ______________________________________

The media used are as follows. YPAD medium contains 1% Bacto YeastExtract, 2% Bacto-Peptone, 2%; Dextrose, 0.003%; and adenine sulfate.Synthetic Dextrose (SD) Medium contains 0.67% Bacto Yeast Nitrogen Basewithout amino acids (Difco), 2% Dextrose and 2% Bacto Agar (Difco).Synthetic Complete (SC) medium is SD medium supplemented with 20 mg eachof adenine, histidine, and uracil, 60 mg of leucine, 30 mg of lysine,and 20 mg of tryptophan per liter of medium. Sporulation medium is 2%Bacto Agar (Difco) and 0.3% potassium acetate. Ura drop-out medium is SCmedium without uracil. Solid media are prepared with approximately 20g/L agar.

EXAMPLE 2

DNA Manipulation and Transformation

Standard techniques of DNA manipulation were utilized (Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a laboratorymanual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). E. coli strain DH5a (Hanahan, D. 1983. Studies on transformationof Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580) was usedas the host in bacterial transformation. Yeast transformation with theDNA libraries was performed by electroporation (Becker, D. and L.Guarente. 1991. High-efficiency transformation of yeast byelectroporation. In C. Guthrie and G. Fink (eds.), Guide to yeastgenetics and molecular biology, Methods Enzymol. 194: 182-187.). Allother yeast transformations with different plasmid subclones were by thealkali cation method (Ito, H., M. Fukuda, M. Murata, and A. Kimura.1983. Transformation of intact yeast cells with alkali cations. J.Bacteriol. 153: 63-68). Plasmid DNA was prepared from E. coli by thealkaline lysis method (Sambrook, J., et al., supra). Plasmids wereisolated from yeast for transformation into E. coli as previouslydescribed (Hoffman, C. S. and F. Winston. 1987. A ten-minute DNApreparation from yeast efficiently releases autonomous plasmids fortransformation of Escherichia coli. Gene 57: 267-272). The wild-typegene, GLS1, which complements the echinocandin-resistance phenotype ofthe MS1 strain was isolated from a yeast genomic DNA library constructedin the centromeric shuttle vector YCp50 (Rose, M. D., P. Novick, J. H.Thomas, D. Botstein, and G. R. Fink. 1987. Gene. 60: 237-243) asdescribed below.

EXAMPLE 3

The nucleotide sequences of the GLS1 5' and the 3' ends were determinedby the dideoxy chain termination method (Sanger, F. S. Nicklen, and A.Coulson. 1977. "DNA sequencing with chain-terminating inhibitors". Proc.Natl. Acad. Sci. USA 74: 5463-5467, 1977), using syntheticoligonucleotide primers complementary to specific regions of GLS1 and asequenase reagent kit (U.S. Biochemical Corp.).

EXAMPLE 4

PCR Amplification

The polymerase chain reactions (PCR) were performed according topublished procedures (Mullis, K. B. and F. A. Faloona, 1987. Specificsynthesis of DNA in vitro via a polymerase-catalyzed chain reaction.Meth. Enzymol. 155: 335-350). Approximately 5 ng of genomic DNA fromstrain X2180-1A was used as a template. Synthetic oligonucleotideprimers were designed to amplify a 1.5-kb fragment of a region 5-kb tothe left of the MAT locus on chromosome III. The sequence of the twoprimers were:

5'-TGACAGTAGTTTCACAAGTACTTAATATTGGAAATG-3' (SEQ ID NO.: 1) and

5'-TCAGATAATTTTATCGGTACCTTTTATATGTTAAAT-3' (SEQ ID NO.: 2).

Amplified DNA fragments were gel-purified, radiolabelled by the randompriming method (Feinberg, A. P and B. Vogelstein. 1984. Anal. Biochem.137: 266) and used as probes to screen bacterial colonies containing ayeast genomic DNA library in the centromeric vector YCp50 (Rose, M. D.,et al., supra) following published protocols (Sambrook, J., et al.,supra).

EXAMPLE 5

Gene Disruption

Chromosomal disruption of the GLS1 gene was performed by one-step genedisruption protocol (Rothstein, R. J. 1983. One-step gene disruption inyeast. Methods Enzymol. 101: 202-211). Plasmid pJAC4 was constructed bycloning of a 1.6-kbp DpnI fragment from pJAC2-1 into the HincII site ofpUC18. Double digestion of pJAC4 with NruI-EcoRV resulted in theexcision of a 1.2-kb fragment containing all but the 40 C-terminal aminoacids encoded by GLS1. A 1.5-kb DNA fragment carrying the S. cerevisiaeURA3 gene was isolated from the YCp50 plasmid as a NruI-SmaI fragment.The purified fragment was treated with T4 DNA polymerase and blunt-endligated into the NruI-EcoRV sites of GLS1 on plasmid pJAC4 resulting inplasmid pJAC9 which contains a disruption deletion of GLS1. PlasmidpJAC9 was propagated in E. coli, and the structure of the plasmid wasverified by restriction analysis.

The gls1::URA3 disruption fragment was purified from pJAC9 as 2.7 kbXbaI-HindIII fragment that was used to transform ura 3-52 yeast strainscontaining a wild-type GLS1 gene (strains GG100-14D and D2-5A). Uracilprototrophic yeast transformants were selected on SC medium lackinguracil. From each of the two transformed strains, one Ura⁺ transformantwas purified by single colony formation. Three Ura⁺ single colonies fromeach of the two transformants were tested for L-733,560-resistance andshown to exhibit drug resistance. These colonies were furthercharacterized and the integration event at the GLS1 locus was confirmedby Southern hybridization (Sambrook, J., et al., supra). The two mutantsresulting from disruption the GLS1 gene in GG100-14D and D2-5A weredesignated MS100 and MS101, respectively.

Total genomic DNA was isolated from stationary cultures of strainsGG100-14D, D2-5A, MS100 and MS101. Approximately 5 μg amounts of eachDNA were restriction digested with DraI. The digestion products wereresolved on 1% agarose gels, followed by transfer to Zeta probe GT nylonmembranes and hybridization according to the manufacturers protocols(Biorad Laboratories). A ³² P radiolabeled probe of a DraI fragment,internal to GLS1, was prepared by the random primer method (Feinberg, A.P and B. Vogelstein. 1984. Anal. Biochem. 137: 266).

EXAMPLE 6

Liquid Broth Microdilution Assay

To quantitate the echinocandin sensitivity/resistance of the mutantstrains, yeast cells grown to log phase were inoculated into 2 ml SCbroth and incubated overnight at 28° C. A flat bottom 96-well microtiterplate was seeded with 75 μl of SC medium in columns 2 through 12. Tocolumn 1, 150 μl medium was added. Echinocandin (L-733,560) was preparedat a concentration of 30 μg/ml solution in sterile distilled water. Analiquot (75 μl) of echinocandin solution was added to column 3, and 75μl from column 3 was transferred to column 4, followed by mixing of thecontents of each well. 75 μl from column 4 was transferred into column5, and the serial dilution was carried on to column 12 from which a 75μl was discarded. A dilution of 6×10⁵ cells/ml in URA drop out mediumwas prepared from the yeast strains. 75 μl cell suspension was thenadded to columns 2 through 12 giving 4.5×10⁴ cells/well. The plates wereincubated at 28° C. for 24-48 hr. Growth in the presence and absence ofechinocandin was measured by absorbance at 600 nm.

EXAMPLE 7

Glucan synthase assay

Membrane extracts were prepared from mutant and wild-type cells grown tologarithmic phase (Kang and Cabib, PNAS, 83, 5808-5812, 1986). Afterhomogenization with glass beads, unbroken cells and debris were removedby low speed centrifugation (1,000×g for 5 min). The supernatant fluidswere centrifuged at 100,000×g for 60 min, and the resulting pellets werewashed with 2.5 ml (per gram of wet cells) of buffer containing 0.05 Mpotassium phosphate (pH 7.5), 0.5 mM DTT, and 1.0 mM PMSF. The washedpellet was resuspended in the same buffer containing 5% glycerol. Thisprotein extract served as the source for both 1,3-β-glucan synthase andthe chitin synthases utilized in the enzymatic assays.

Protein concentrations were determined using BCA protein assay reagentkit (Pierce Corp.), utilizing bovine serum album (BSA; Pierce Corp.) asa standard. The 1,3-β-glucan synthase reactions were performed aspreviously described (Cabib, E., and M. S. Kang. 1987. Fungal1,3-b-glucan synthase, Methods Enzymol. 138: 637-642). Briefly, an 80 mlreaction contained 125 mM Tris HCL (pH7.5), 0.25 mM dithiothretol, 30 mMKF, 0.3 M glycerol, 0.23% BSA, 0.125 mM PMSF, 2 mM UDP-glucose, 10 mMguanosine 5'-(g-thio) triphosphate (GTPγS), 0.1 nmol UDP- ³ H! glucose(Amersham; 4.5 Ci/mmol;) plus 25 mg of membrane protein extract. Thereactions were performed in the presence of 0.0, 0.1, 0.5, 5, 25 and 50mM L, 733-560 for dose titration of the drug. Following incubation at25° C. for 150 minutes, the ³ H!-glucose incorporated intotrichloroacetic acid-insoluble material was collected onto glass fiberfilters (102×258 mm) and measured using a betaplate liquid scintillationcounter (Cambridge Technologies Inc.; series 2800 harvester) at 25%efficiency. The product of such reactions was verified by solubilizationby laminarinase (Sigma, #L9259) but not by α-amylase (Sigma, #A2643).

EXAMPLE 8

Chitin synthase assay

A previously described chitin synthase assay was used (Kang, M. S., N.Elango, E. Mattia, J. Au-Young, P. W. Robbins, and E. Cabib. 1984.Isolation of chitin synthetase from Saccharomyces cerevisiae.Purification of an enzyme by entrapment in the reaction product. J.Biol. Chem. 259: 14966-14972). Approximately 125 mg of membrane proteinextract was trypsin-activated and used to catalyze chitin synthasereactions. A reaction of 100 ml contained 50 mM Tris HCl, pH 7.5, 40 mMMgCl, 32 mM N-acetylglucosamine (GlcNAc), 1 mM UDP-N-acetyl- ¹⁴C!glucosamine (4×10⁵ cpm/umol), 0.8 mg/ml digitonin. The reactions wereperformed in the presence of 0.0, 0.125, 0.5, 2 and 8 μM of nikkomycinZ. After 30 minutes of incubation at 30° C., the reaction products wereprecipitated with 10% trichloroacetic acid and collected onto Whatmanglass microfiber GF/A discs followed by counting of the incorporated ¹⁴C!-GlcNAc.

EXAMPLE 9

Isolation of Spontaneous Echinocandin-Resistant Mutants

To isolate mutants in genes involved in biosynthesis of 1,3-β-glucan,pneumocandin B compound, L-733,560 was used to identify resistantmutants in strain X2180-1A. Approximately 40 spontaneous mutants capableof colony formation in the presence of 7.5 mM of L-733,560 were isolatedas follows: wild-type strain X2180-1A was grown to stationary phase inSD minimal medium. Approximately 1-3×10⁶ cells were spread on SD platescontaining 7.5, 15 or 45 mM L-733,560. Following incubation at 28° C.for four days, echinocandin-resistant colonies appeared at a frequencyof 1-3×10⁶. Mutant strains MS1, MS41 and MS14 were isolated by thisprocedure. MS14 contains a mutation (fks1-4) in the FKS1 gene. MS1 andMS41 contain gls1-1 and gls1-2 mutations, respectively.

EXAMPLE 10

EMS Mutagenesis and Isolation of Strain MS43 Carrying the gls1-3mutation

YPAD broth (5 ml) was inoculated with an overnight culture of strainX2180-1A to give initial cell density of 1×10⁶ cell per ml and incubatedat 30° C. overnight. A 2.5 ml aliquot of the overnight culture waswashed twice in 50 mM KPO₄ buffer, pH 7.0 by centrifugation, andresuspended in 10 ml of the same buffer. To a 5 ml aliquot of the washedcells, 150 μl of ethyl methanesulfonate (EMS) was added. The suspensionof treated cells was vortexed and incubated at 30° C. for 1 hr. Theother 5 ml of the washed culture was kept on ice untreated. To the 5 mltreated cells, an equal volume of a freshly prepared 10% (w/v)filter-sterilized sodium thiosulfate solution was added and mixed. Cellswere collected, washed twice with sterile water, resuspended in 5 mlYPAD, and incubated at 24° C. for 4-6 hr. Appropriate cell dilutionswere plated on minimal medium (SD). The untreated culture was dilutedand plated by the same way. After 3 days of growth at 24° C., colonieswere replica-plated onto YPAD medium with and without 0.001 μg/mlL-733,560 and incubated at 30° C. Of 1000 colonies tested by thisprocedure, 10 colonies were resistant to L-733,560. One of theseresistant strains, designated MS43, was shown to contain the gls1-3mutation as described below.

EXAMPLE 11

Genetic Analysis

Outcrosses were performed between each of three mutant strains (MS1,MS41, MS43) and the wild-type strain GG100-14D. Tetrad analysis revealedthat L-733,560-resistance segregated as a single trait in all threemutant strains (FIG. 1). Single gene mutations are expected to segregatein a mendelian fashion (2:2) upon crossing of a mutant to a wild-typestrain.

The mutations in the three mutant strains were tested for dominance orrecessiveness by mating the MATa echinocandin-resistant mutants to theMATa echinocandin-sensitive strain GG100-14D (wild-type). All threeresulting MATa/MATa heterozygous diploid strains, D2(MS1xGG100-14D), D12(MS41xGG100-14D and D28 (MS43xGG100-14D), exhibited sensitivity toL-733,560, indicating that strains MS1, MS41 and MS43 contain recessivemutations.

The recessive nature of these mutations was verified by the finding thatthe heterozygous diploids D2, D12 and D28 exhibited the wild-type ratherthan the mutant phenotype. Complementation tests were performed usingdrug-resistant segregants from the D2, D12 and D28 diploids. Thediploids formed between resistant isolates carrying mutations from MS1,MS41 or MS43 (D132, D136, D137, D140, D141 and D142) exhibitedresistance to L-733,560, indicating lack of complementation among thethree mutations.

These results indicate that the three independently isolated mutationsgls1-1 (strain MS1), gls1-2 (strain MS41) and gls1 -3 (MS43) compriseone complementation group. Mutations in different or unlinked genes cancomplement a specific phenotype. Mutations in the same gene or intightly-linked genes usually fail to complement each other and are,therefore, classified as one complementation group.

EXAMPLE 12

Genetic Mapping of the gls1 Mutations

Genetic analysis of the meiotic segregants of 37 tetrads resulting fromoutcrossing MS41 to GG100-14D lead to mapping of the gls1-1 mutation towithin 1.35 centi Morgan from the MAT locus on chromosome III (Table 2).Similar analysis revealed that both gls1-2 (strain MS41) and and gls1-3(strain MS43) are linked to the MAT locus. The parental ditype class oftetrads (PD) was the only class obtained from crosses between GG100-14Dand MS1(12 tetrads) or between GG100-14-D and MES43 (19 tetrads). If themutations were in unlinked loci or in genes located on differentchromosomes, then a predominant tetratype class (T) of progeny wouldhave been expected. The phenotypes of the mutant and the wild-typestrain used in the genetic crosses are given below:

    ______________________________________    Strain            Phenotype    ______________________________________    MS1               Mat a, Echinocandin.sup.R    MS41              Mat a, Echinocandin.sup.R    MS43              Mat a, Echinocandin.sup.R    GG100-14D         Mat a Echinocandin.sup.S    ______________________________________

The tetrads resulting from outcrossing a MATa resistant mutant (strainsMS1, MS41 or MS43) to a MATa sensitive wild-type (GG100-14D) shouldexhibit one of the three tetrad types as follows:

1. Parental ditype phenotypes (PD): Mat a Echinocandin^(R)

Mat a Echinocandin^(S)

The siblings exhibit the phenotypes of either parent.

2. Non-parental ditvpe phenotypes (NPD): Mat a Echinocandin^(S)

Mat a Echinocandin^(R)

The siblings exhibit phenotypes of neither parent.

3. Tetratype phenotypes (T): The siblings exhibit both parental andnon-parental ditype phenotypes (Mat a Echinocandin^(S), Mat aEchinocandin^(R), Mat a Echinocandin^(R), Mat a Echinocandin^(S).

The data obtained from outcrossing the three gls1 mutants to thewild-type strain GG100-14D is summarized in Table 2.

                  TABLE 2    ______________________________________    GENETIC MAPPING OF THE GLS1 MUTATIONS              Ascus Type       Map Distance    Interval    PD    NPD        T   (cM)    ______________________________________    gls1-1-MAT  36    0          1   1.35    gls1-2-MAT  12    0          0    gls1-3-MAT  19    0          0    ______________________________________

A NPD ratio of <1 indicates linkage. This data indicates that the threegls1 mutations are linked to MAT. The map distance between two loci iscalculated as follows: 100 (1/2T)+NPD/ Total number of tetrads Thedistance between gls1-1 and MAT=50/37=1.35 centi Morgan.

EXAMPLE 13

Characterization of Strain MS1 Mutant

Strain MS1 exhibits a bilateral mating defect, (i.e., mating for theproduction of homozygous diploids containing two copies of gls1-1 isvery ineffecient). The resultant homozygous diploids do not form sporesupon subculturing on sporulation media. The homozygous diploids areosmotically unstable and burst when suspended in water.

In contrast, mating between wild-type cells and cells containing any ofthe three gls1 mutations for production of heterozygous diploids isnormal.

Morphologically, MS1 cells show some aggregated cells (FIG. 3), multiplebuds, and occasional flocculated growth in YPAD medium at 30° C. StrainMS1 also grows slower in YPAD than its wild-type parental strain. Thisslow growth is characterized by a long lag period before the cells enterthe division cycle.

EXAMPLE 14

Effect of Antifungal Drugs on Strain MS1

Strain MS1 did not exhibit multiple drug resistance when tested againsta panel of more than 30 inhibitors affecting cell wall, membrane,sterol, and protein synthesis.

In addition to its resistance to L-733,560 (FIG. 2), MS1 cells are moresensitive to the 1,3-β-glucan synthase inhibitor, papulacandin, and tothe immunosupressant rapamycin, than is the wild-type parental strain(FIG. 10).

EXAMPLE 15

Levels of 1,3-β-Glucan Synthase and Chitin Synthase Activities

Crude enzyme preparations from cell membranes were tested for 1,3-βglucan synthase and chitin synthase activities. The sensitivity of invitro synthesis of those polymers to L-733,560 and Nikkomycin Z wasdetermined (FIG. 4).

These experiments showed that glucan synthesis activity of MS1 isreduced (80% less activity) relative to wild-type cells.

EXAMPLE 16

Stimulation of 1,3-β-D-Glucan Synthesis by GTPγS

To study activation of 1,3-β-glucan synthesis by GTPγS incorporation ofUDP-glucose into 1,3-β-D glucans, in absence and in presence of 3.3 uMGTPγS was measured. The glucan synthesis of the MS1 mutant enzymaticactivities was stimulated (approximately 19-fold) by GTPγS. Thiscontrasts the 6-fold stimulation by the MS1 mutant enzyme (FIG. 4).

In another experiment the stimulation of the mutant and the wild-typemembranes by different concentrations of GTPγS was studied. The MS1mutant enzymatic activity responded rather poorly to increasingconcentrations of GTPγS (FIG. 5).

EXAMPLE 17

Isolation of the GLS1 Gene by Functional Complementation and byHybridization

The GLS1 gene was cloned by complementation of theechinocandin-resistance phenotype. Yeast strain D2.8B (MAT a, ura3-52,gls1-1) was transformed with a yeast genomic DNA library in thecentromeric vector YcP50 (Rose, M. D., et al., 1987. Gene. 60: 237-243),followed by selection of transformants on Ura-drop-out medium.Transformants (2400) were picked onto master plates of Ura-drop outmedium and replica-plated onto plates containing the same mediumsupplemented with 0.0 or 7.5 μM of L-733-560. Following incubation at30° C. for 2-3 days, 4 sensitive colonies colonies were isolated. One ofthese four colonies, designated D2.8B (pJAC2- 1) was shown to contain acomplementing plasmid, pJAC2-1. In a separate experiment, a 1.5 kb DNAfragment representing a sequence located at about 7 kb to the left ofMAT was amplified by PCR, radiolabeled and used as a probe to screen aYCp50-based yeast genomic library by colony hybridization (Sambrook, J.,et al., supra). Screening of approximately 4800 bacterial colonies bythis procedure resulted in a hybridizing clone containing a plasmiddesignated pJAC2-2.

pJAC2-1 and pJAC2-2 contain DNA fragments that have identicalrestriction maps. pJAC2-2 was introduced into strain D2.8B. Three yeasttransformants were tested and shown to have acquired a wild-type levelof sensitivity to L-733-560. Furthermore, membrane extracts preparedfrom the transformants reversed the low level of 1,3-β-glucan synthasespecific activity associated with the membranes of the untransformedmutant.

A yeast transformant designated D2.8B (pJAC2-2) was cured of itstransforming plasmid, pJAC2-2, by three successive rounds of overnightgrowth in YPAD broth followed by plating on YPAD plates for singlecolony formation. Cured clones lost the plasmid and exhibited resistanceto L-733,560.

EXAMPLE 18

Determination of the Gls1-1 Minimum Complementing Fragment

The gls1-1 complementing region of pJAC2 was defined by digesting withrestriction enzymes that cut within the cloned insert DNA (FIG. 6).Plasmids containing restriction fragments subcloned in YcP50 werepropagated in E. coli DH5a, characterized with regard to restrictionpatterns and then introduced into mutant yeast strain MSD2.8B. Theresulting yeast transformants were tested for growth rate anddrug-resistance phenotype. The gls1-1 complementing activity was presentin approximately 4 kb KpnI fragment (FIG. 1). Further complementationanalysis defined a 1.6 kbp DpnI fragment containing the GLS1 gene.

Using restriction analysis and subcloning of smaller DNA fragments fromthe original 17 kb library clone, a 4 kb KpnI fragment of pJAC2 wascloned in the single copy vector YCpLac33 to yield pJAC 1. pJAC 1complemented the MS1 mutant phenotypes.

By similar analysis a 1.6 kb DpnI fragment from pJAC2 was cloned intothe HincII site of the bacterial vector pUC 18 to produce a plasmiddesignated pJAC4. A BamHI/SphI 1.6 kb fragment containing GLS1 waspurified from pJAC4 and subcloned into both YEp24 and YCp50 plasmids(digested with BamH1 and SPH1) to yield pJAC5 and pJAC3 respectively.Both plasmids (pJAC3 and pJAC5) complemented the gls1-1drug-resistance/sensitivity phenotypes (FIGS. 7, 8).

EXAMPLE 19

Nucleotide and Deduced Amino Acid Sequence Analysis of GLS1

The dideoxy chain termination method was used to determine thenucleotide sequence of the 5' end of GLS1. The sequence was determinedfor the first 200 bp and compared with the open reading frame (ORF)YCR34 on Chromosome III (Olwer, S. et al., 1992, Nature 357: 38-46). Thesequences were identical over the 200 bp compared. These resultssuggested the identity of GLS1 and the YCR34 ORF. The nucleotide (SEQ IDNO: 1) and the predicted amino acid (SEQ ID NO: 2) sequences ofGLS1/YCR34 are shown in FIG. 9.

The 600 bp of sequence in the 5' untranslated region of GLS1/YCR34contains four candidate promoter `TATA` boxes. In addition, there aretwo candidate UAS elements with a strong homology to the yeast HAP1binding site. Thus, the promoter region of GLS1 shows a strikingsimilarity to that of the yeast gene CYC1. The sequence matches of the 4`TATA` elements are almost identical in the two genes. The UAS sequencesalso show a strong homology to the UAS1 of CYC1. This suggests that GLS1expression may be controlled in a fashion similar to that of CYC1.

The 348 amino acid putative protein product of GLS1/YCR534 was comparedto protein databases. No significant homology with known proteins wasfound. A hydropathy analysis was performed on the 348-residue amino acidsequence, using the Kyte and Doolittle algorithm (FIG. 10). The putativeprotein product is basic (pI=10.3) and hydrophobic. There are severalleucine zipper motifs in the sequence, indicating that the proteinproduct may fold as a dimer.

EXAMPLE 20

GLS1 Gene Disruption

A chromosomal deletion of the GLS1 gene was generated by one-step genedisruption (Rothstein, R. J., 1983, Methods Enzymol. 1012: 202-211) totest whether GLS1 is essential. A 1.2 kb region of plasmid pJAC4,containing most of the GLS1 coding sequence, was deleted as a NruI-EcoRVfragment. The deleted region of pJAC4 was replaced by blunt-end ligationwith a 1.5 kb DNA fragment containing the URA3 gene. The disrupted copyof GLS1 was excised as a 2.7-kbp HindII/XbaI fragment, and used totransform the two GLS1-containing wild-type strains GG100-14D and D2.5A.

The resulting Ura⁺ yeast transformants were tested forechinocandin-resistance. Two transformants (GGDgls1 and D2.5ADgls1)acquired resistance to L-733,560 and were analyzed further (FIG. 9). Thealteration of the GLS1 locus was confirmed by Southern hybridizationanalysis. The viability of the haploid strains with GLS1 deletionsindicate that the gene is not essential for growth.

EXAMPLE 21

GLS1 Homologues Exist in Pathogenic Species

Yeast 1,3-β-D glucan synthase can be fractionated into a soluble andinsoluble fractions by treating yeast membrane preparations with saltand detergent. A glucan synthase activity can be reconstituted by mixingthe two fractions in presence of GTP. Cabib and coworkers havedemonstrated that the solubilized fraction is exchangeable between yeastand other fungi. This suggests a possible homology between glucansynthesis enzymes amongst fungi.

To test whether homologues of the cloned GLS1 exist in other fungi,genomic DNA from several heterologous species was prepared and a seriesof PCR and Southern hybridization analysis were performed. The resultsshowed that GLS1 homologues exists in other fungi, including Candidaalbicans, Aspergillus fumigatus, Schizosaccharomyces pombe, andPhytophthora infestans (FIG. 11).

EXAMPLE 22

Isolation of GLS1 Homologues from Pneumocystis Carinii

Whole rat lungs from P. carinii -infected male Sprague-Dawley rats arehomogenized with a Brinkmann homogenizer, and DNA is isolated asdescribed (P. A. Liberator, et al., 1992. J. Clin. Micro. 30(11):2968-2974). Two to five micrograms of purified DNA are digested with arestriction endonuclease such as EcoRI, and the fragments are separatedon an agarose gel. DNA is transferred to a solid support such asnitrocellulose and probed by the method of Southern (Southern, E. M.1975. J. Mol. Biol. 98: 503-517) for fragments with homology to GLS1. Bywashing the blot at a reduced stringency, weakly homologous genes can beidentified.

The P. carinii GLS1 homologues are cloned by preparing a mini-libraryfrom the region of the agarose gel where the hybridizing fragment wasvisualized on the Southern blot. Following phenol:CHCl₃ extraction toremove contaminants, DNA fragments from this area of the gel are ligatedinto an appropriate plasmid vector and transformed into E. coli. The E.coli clones bearing the mini-library are spread onto agar plates andprobed for inserts homologous to GLS1 by in situ colony lysis. DNA fromindividual transformants is transferred to nitrocellulose, hybridized toa radiolabeled GLS1 DNA fragment, washed, and exposed to film. Coloniescontaining an insert with homology to GLS1 are visualized on the film;plasmid DNA is then isolated from positive clones, propagated, andanalyzed. DNA sequence analysis by standard methods is used to establishthe extent of homology to GLS1, and functional homology may bedemonstrated by expression in S. cerevisiae disrupted for GLS1.

EXAMPLE 23

Isolation of GLS1 Homologs from Phytopathogenic Fungi

To clone GLS1 homologs from phytopathogenic fungi such as Phytophthorainfestans, high molecular weight genomic DNA is isolated by the methoddescribed by Atkins and Lambowitz (Mol. Cell. Biol., 5; 2272-2278),partially digested by a restriction enzyme, and cloned into theStratagene Vector Lambda-Dash using a cloning kit obtained from themanufacturer and methods of the art (Maniatis). The libraries arescreened using probes from GLS1.

EXAMPLE 24

The GLS1 mutants (strains MS1, MS41 and MES7-43) areechinocandin-resistant and papulacandin-supersensitive, while the fks1-4mutant (strain MS14) is echinocandin-resistant (50 fold more resistantthan wild-type) and nikkomycin Z-supersensitive (1000 fold moresensitive). The GLS1 and the fks1-4 mutants can be incorporated into anassay to screen and classify antifungal compounds with chitin and glucansynthase inhibitory effects, based on their differentialresistance/sensitivity to the echinocandins, papulacandin and NikkomycinZ. The data from this assay and the sizes of the zones of growthinhibition in millimeter is given in the following table:

    ______________________________________                  Strain                        X2180      MS1   MS14    Inhibitor   μg/Disc                        (WT)       (gls1-1)                                         (fks1-4)    ______________________________________    L, 733-560  20      15         12    8 vh    (echinocandin)    L-688-786   10      0          7 vh  0.0    (echinocandin)    Aculeacin   50      15         9     0.0    Papulacandin                50      10         20    10 h    Nikkomycin Z                10      0.0        0.0   30.0    Rapamycm    12.5    0.0        13    0.0    ______________________________________

The MS1 and MS14 yeast strains may be used in an assay to screen forglucan and chitin synthesis inhibitors. This assay can also discriminatebetween different classes of glucan synthesis inhibitors likepapulacandins and echinocandins.

A compound that is active against MS14 but inactive against MS1 and thewild-type strain is a "chitin synthase-type" of inhibitor. A compoundthat is active against MS1 but less active against the wild-type strainand MS14 is a "papulacandin type" of inhibitor. "Echinocandin-type"inhibitors would exhibit less activity on MS1 cells and lesser activityon MS14 cells relative to the wild-type strain.

EXAMPLE 25

Cloning of GLS1 for Expression of the GLS1 Polypeptide In Other HostCell Systems

(a) Cloning of GLS1 cDNA into a bacterial expression vector. RecombinantGLS1 is produced in a bacterium such as E. coli following the insertionof the optimal GLS1 cDNA sequence into expression vectors designed todirect the expression of heterologous proteins. These vectors areconstructed such that recombinant GLS1 is synthesized alone or as afusion protein for subsequent manipulation. Expression may be controlledsuch that recombinant GLS1 is recovered as a soluble protein or withininsoluble inclusion bodies. Vectors such as pBR322, pSKF, pUR, pATH,pGEX, pT7-5, pT7-6, pT7-7, pET, pIBI (IBI), pSP6/T7-19 (Gibco/BRL),pBluescript II (Stratagene), pTZ18R, pTZ19R (USB), pSE420 (Invitrogen)or the like are suitable for these purposes.

(b) Cloning of GLS1 cDNA into a viral expression vector. RecombinantGLS1 is produced in mammalian host cells, such as HeLa S3 cells, afterinfection with vaccinia virus containing the GLS1 cDNA sequence. Toproduce GLS1:vaccinia virus, the GLS1 cDNA is first ligated into atransfer vector, such as pSC11, pTKgptF1s, pMJ601 or other suitablevector, then transferred to vaccinia virus by homologous recombination.After plaque purification and virus amplification, GLS1:vaccinia virusis used to infect mammalian host cells and produce recombinant GLS1protein.

EXAMPLE 26

Process for the Production of a Glucan Synthase Subunit Peptide

Recombinant GLS1 is produced by (a) transforming a host cell with DNAencoding GLS1 protein to produce a recombinant host cell; (b) culturingthe recombinant host cell under conditions which allow the production ofglucan synthase subunit peptide; and (c) recovering the recombinantglucan synthase subunit peptide. The recombinant glucan synthase subunitis purified and characterized by standard methods.

EXAMPLE 27

Compounds that modulate glucan synthase subunit activity may be detectedby a variety of methods. A method of identifying compounds that affectglucan synthase subunit comprises:

(a) mixing a test compound with a solution containing glucan synthasesubunit to form a mixture;

(b) measuring glucan synthase subunit activity in the mixture; and

(c) comparing the glucan synthase subunit activity of the mixture to astandard.

Compounds that modulate glucan synthase subunit activity may beformulated into pharmaceutical compositions. Such pharmaceuticalcompositions may be useful for treating diseases or conditions that arecharacterized by fungal infection.

EXAMPLE 28

DNA which is structurally related to DNA encoding glucan synthasesubunit is detected with a probe. A suitable probe may be derived fromDNA having all or a portion of the nucleotide sequence of the figures,RNA encoded by DNA having all or a portion of the nucleotide sequence offigures, degenerate oligonucleotides derived from a portion of the aminoacid sequence of figures or an antibody directed against the peptideencoded by GLS1.

EXAMPLE 29

A kit useful for the detection and characterization of DNA or RNAencoding glucan synthase subunit or glucan synthase subunit peptide isprepared by conventional methods. The kit may contain DNA encodingglucan synthase subunit, recombinant glucan synthase subunit peptide,RNA corresponding to the DNA encoding glucan synthase subunit orantibodies to glucan synthase subunit. The kit may be used tocharacterize test samples, such as forensic samples or epidemiologicalsamples.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 2    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1854 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    - ATTTCAGCAT GCTATTTCTC AAGGCACTCC TACTTTCCCT TTACCGGCCC CT - #CGCACTAG      60    - TCCAATAAGT CGTGCGCCTC CAAAGTTCAA TTTTTCGAAT GATCCGTTGG CA - #GCTTTGGC     120    - TGCGGTTGCC TCCGCGCCAG ATGCAATGAG CAGTTTTTTA TCTAAAAAGG AA - #AATAATAA     180    - TTGAACAAAC GGCTGAGACG GGCAATACAT ATGCTCTACT TCTTTTCCAT CC - #AATGGTTG     240    - GTGAAACTCT CGAGCATACA TTACCTTACG TGTGTTAGTG TACTATATTA TA - #TATATATA     300    - TATGTATATA TATAAAGGGA GGAGTTTTTA ATTATAATTG TAATTTCGTA TT - #TTTTCTGC     360    - ATTATACAGT TTTTTCCGAT TTTAAACGAC TTTATTTAAG TGTCGTGTAA AT - #ATGTCACA     420    - TTTTATTTTT GTACGTATTC ACATGTCCTG GCGTGCGGCC ATTGCTGAAA AT - #CGCAAAAC     480    - CCACAGAGAA ATAAACATCG CGAAAAAGTC AATGAAAAAT TGGAAAATAT TT - #TTCATTTC     540    - ACTATTATCC ACAAGCAATT TTGTACAAAG TGAAAAGGTT GAACTAATTA TC - #TTCGTCTA     600    - GAAGCCATGA ATTCACTCGT TACTCAATAT GCTGCTCCGT TGTTCGAGCG TT - #ATCCCCAA     660    - CTTCATGACT ATTTACCAAC TTTGGAGCGA CCATTTTTTA ATATTTCGTT GT - #GGGAACAT     720    - TTCGATGATG TCGTCACTCG TGTAACTAAC GGTAGATTTG TTCCAAGCGA AT - #TCCAATTC     780    - ATTGCAGGTG AATTACCATT AAGCACTTTG CCCCCTGTGC TATACGCCAT CA - #CTGCCTAT     840    - TACGTTATTA TTTTTGGTGG CAGGTTTTTG TTAAGTAAGT CGAAACCATT TA - #AATTAAAT     900    - GGCCTTTTCC AATTGCATAA TTTGGTTTTA ACTTCACTTT CATTGACGCT TT - #TATTGCTT     960    - ATGGTTGAAC AATTAGTGCC AATTATTGTT CAGCACGGGT TATACTTCGC TA - #TCTGTAAT    1020    - ATTGGTGCTT GGACTCAACC GCTCGTTACA TTATATTACA TGAATTACAT TG - #TCAAGTTT    1080    - ATTGAATTTA TAGACACCTT TTTCTTGGTG CTAAAACATA AAAAATTGAC AT - #TTTTGCAT    1140    - ACTTATCACC ATGGCGCTAC TGCCTTATTA TGTTACACCC AATTGATGGG CA - #CCACATCT    1200    - ATTTCTTGGG TCCCTATTTC ATTGAACCTT GGTGTTCACG TGGTTATGTA TT - #GGTACTAT    1260    - TTCTTGGCTG CCAGAGGCAT CAGGGTCTGG TGGAAGGAAT GGGTTACCAG AT - #TTCAAATT    1320    - ATCCAATTTG TTTTGGATAT CGGTTTCATA TATTTTGCTG TCTACCAAAA AG - #CAGTTCAC    1380    - TTGTATTTCC CAATTTTGCC ACATTGTGGT GACTGTGTGG GTTCAACAAC TG - #CCACCTTT    1440    - GCAGGTTGTG CCATTATTTC TTCATATTTG GTACTATTTA TTTCATTTTA CA - #TTAACGTT    1500    - TATAAACGTA AAGGCACCAA AACCAGTAGA GTGGTAAAGC GTGCCCACGG CG - #GTGTTGCC    1560    - GCAAAGGTTA ATGAGTATGT TAACGTTGAC TTGAAAAACG TTCCTACTCC AT - #CTCCATCA    1620    - CCAAAACCTC AACACAGAAG AAAAAGGTAA GTGTAAAATC TTTGAAAGAA TT - #TAAGTATT    1680    - CAACTTTCGT ATATTCGTTT TTTCTTAGTG GATCTATTGT TACTATTATC AC - #TATTATTA    1740    - TATTGTAAAA GACCGGATGG TTTTGTTATA TATTACATAC ACATGTTATC GT - #TGAAAAAA    1800    - GTTTTCCGTT TCCTTTCGAC AGTCATCAGA TAATTTTATC CGAGTCTTTT AT - #AT    1854    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 347 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Asn Ser Leu Val Thr Gln Tyr - # Ala Ala Pro Leu Phe Glu Arg    Tyr    #   15    -      Pro Gln Leu His Asp Tyr Leu Pro - # Thr Leu Glu Arg Pro Phe Phe    Asn    #                 30    -      Ile Ser Leu Trp Glu His Phe Asp - # Asp Val Val Thr Arg Val Thr    Asn    #             45    -      Gly Arg Phe Val Pro Ser Glu Phe - # Gln Phe Ile Ala Gly Glu Leu    Pro    #         60    -      Leu Ser Thr Leu Pro Pro Val Leu - # Tyr Ala Ile Thr Ala Tyr Tyr    Val    #     80    -      Ile Ile Phe Gly Gly Arg Phe Leu - # Leu Ser Lys Ser Lys Pro Phe    Lys    #   95    -      Leu Asn Gly Leu Phe Gln Leu His - # Asn Leu Val Leu Thr Ser Leu    Ser    #                110    -      Leu Thr Leu Leu Leu Leu Met Val - # Glu Gln Leu Val Pro Ile Ile    Val    #            125    -      Gln His Gly Leu Tyr Phe Ala Ile - # Cys Asn Ile Gly Ala Trp Thr    Gln    #        140    -      Pro Leu Val Thr Leu Tyr Tyr Met - # Asn Tyr Ile Val Lys Phe Ile    Glu    #    160    -      Phe Ile Asp Thr Phe Phe Leu Val - # Leu Lys His Lys Lys Leu Thr    Phe    #   175    -      Leu His Thr Tyr His His Gly Ala - # Thr Ala Leu Leu Cys Tyr Thr    Gln    #                190    -      Leu Met Gly Thr Thr Ser Ile Ser - # Trp Val Pro Ile Ser Leu Asn    Leu    #            205    -      Gly Val His Val Val Met Tyr Trp - # Tyr Tyr Phe Leu Ala Ala Arg    Gly    #        220    -      Ile Arg Val Trp Trp Lys Glu Trp - # Val Thr Arg Phe Gln Ile Ile    Gln    #    240    -      Phe Val Leu Asp Ile Gly Phe Ile - # Tyr Phe Ala Val Tyr Gln Lys    Ala    #   255    -      Val His Leu Tyr Phe Pro Ile Leu - # Pro His Cys Gly Asp Cys Val    Gly    #                270    -      Ser Thr Thr Ala Thr Phe Ala Gly - # Cys Ala Ile Ile Ser Ser Tyr    Leu    #            285    -      Val Leu Phe Ile Ser Phe Tyr Ile - # Asn Val Tyr Lys Arg Lys Gly    Thr    #        300    -      Lys Thr Ser Arg Val Val Lys Arg - # Ala His Gly Gly Val Ala Ala    Lys    #    320    -      Val Asn Glu Tyr Val Asn Val Asp - # Leu Lys Asn Val Pro Thr Pro    Ser    #   335    -      Pro Ser Pro Lys Pro Gln His Arg - # Arg Lys Arg    #                345    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified glucan synthase subunitpeptide having the amino acid sequence of SEQ ID NO: 2.